Molecular Interactions within the Niche

Adhesion is perhaps the most obvious interaction between the stem cell and the niche. HSC retention in the vascular niche is mediated in part by CXCL12 (SDF-1), which is also important in homing and mobilization. Several cell types in the niche express CXCL12, including osteoblasts and vascular endothelial cells. The binding of CXCL12 to its receptor, CXCR4, located on HSCs, is a crucial interaction in the retention and maintenance of adult HSCs (Wilson and Trump 2006). CXCL12 is regulated by fibroblast growth factor 2 (FGF2) that binds one of the fibroblast growth factor receptors (FGFR1 iiic) located on stromal cells of the niche. The binding of FGF2 to FGFRs causes an increase in the degradation of CXCL12 mRNA, reducing its secretion into the extracellular space of the niche. FGF2 can thereby negatively regulate the retention of HSCs in the niche and influence the maintenance of hematopoiesis (Nakayama et al. 2007). Many other adhesion molecules are important in the HSC-niche interaction (Fig. 6.3). VLA4 on HSCs binds vascular cell-adhesion molecule 1 (VCAM1) on sinusoidal endothelial cells. CD44 on HSC membranes binds hyaluronic acid that is present in the niche microenvironment (Wilson and Trump 2006). Another important regulatory molecule in the niche is bone morphogenic protein receptor 1A (BMPR1A), which, as mentioned previously, is normally expressed on osteoblasts lining the endosteal niche (Wilson and Trump 2006). Mice that lack BMPR1A in the stroma display an increase in the number of osteoblasts and repopulating HSCs. Direct contact between HSCs and stromal osteoblasts is also mediated by interactions with N-cadherin, specifically through a group of specialized spindle-shaped N-cadherin-expressing osteoblasts termed "SNOs" (Zhang et al. 2003). In addition to SNOs, HSCs may also express N-cadherin when stimulated by angiopoietin-1 (Angl). Angl, which is secreted by osteoblasts, stimulates Tie2 on HSCs, ultimately influencing cell cycle regulation via the phosphoinositide 3-kinase-Akt-p21 pathway. This interaction blocks cell division and mediates adhesion of the HSC to the niche by increasing

Fig. 6.3 HSCs engage in a number of cell-cell and cell-matrix interactions in the niche. Various cells of the niche (such as osteoblasts and stromal cells) have been shown to express integrins and signaling receptors on the cell surface, and secrete factors such as OPN, SCF, and CXCL12 that have binding partners on HSCs. HSCs also interact with extracellular matrix molecules such as hyaluronic acid via the CD44 molecule on the cell surface. Each of these molecular interactions has been shown to influence HSC functions such as adhesion, proliferation and quiescence (See Color Plate)

Fig. 6.3 HSCs engage in a number of cell-cell and cell-matrix interactions in the niche. Various cells of the niche (such as osteoblasts and stromal cells) have been shown to express integrins and signaling receptors on the cell surface, and secrete factors such as OPN, SCF, and CXCL12 that have binding partners on HSCs. HSCs also interact with extracellular matrix molecules such as hyaluronic acid via the CD44 molecule on the cell surface. Each of these molecular interactions has been shown to influence HSC functions such as adhesion, proliferation and quiescence (See Color Plate)

Niche Cell

Niche Cell

HSC N-cadherin expression, thus contributing to HSC quiescence and attachment to the niche. These pathways intimately link HSC adhesion to the niche with cell cycle regulation (Arai et al. 2004). Osteoblasts can also influence the number of HSCs in the bone marrow through the secretion of osteopontin (OPN) into the bone matrix. OPN is a glycoprotein that binds to CD44 on HSC membranes (Sugiyama et al. 2006). OPN-deficient mice exhibit a twofold increase in HSCs, suggesting that secretion of OPN by osteoblasts is a mechanism by which the endosteal niche negatively regulates HSC number (Nilsson et al. 2005, Stier et al. 2005).

Another regulator of stem cell frequency is the Wnt pathway, which is activated by the binding of the Wnt protein (from the niche) to its cell-surface receptor complex, frizzled (on the stem cell membrane). Absence of the Wnt-frizzled interaction results in degradation of P-catenin by the proteasome. When Wnt is bound to frizzled this degradation is blocked, and P-catenin accumulates and translocates to the nucleus, resulting in the activation of several target genes. (For a more comprehensive review of this signaling pathway, see Clevers 2006. In the hematopoietic system, activation of the Wnt signaling pathway results in HSC proliferation and inhibition of differentiation, effectively increasing the stem cell pool (Reya and Clevers 2005, Staal and Clevers 2005).

The Notch signaling pathway is a regulator of cell fate in a wide range of developmental processes and cellular systems. Notch is a cell surface receptor located on HSCs that binds the transmembrane protein ligand, Jaggedl, located on cells of the endosteal niche. This binding results in the cleavage of the intracellular domain of the notch receptor, which then translocates to the nucleus and activates notch target genes (Kojika and Griffin 2001, Ross and Li 2006). This signaling cascade in HSCs mediates cell fate determination and self-renewal in the endosteal niche. Osteoblasts produce high levels of Jaggedl to increase the frequency of HSCs in the niche via the Notch signaling pathway (Calvi et al. 2003). Integration of the Wnt and Notch signaling pathways contributes to tight regulation of the frequency of HSCs. Notch/ Wnt double-reporter mice were used to determine that most cells in the stem cell niche use both pathways at the same time and stimulation of the Wnt pathway causes a transcriptional increase in Notch targets (Duncan et al. 2005).

Another important interaction between the niche and stem cells involves stem cell factor (SCF), which is encoded by the steel (Sl) locus and is present in both a membrane-bound (on osteoblasts) and secreted form. SCF binds and activates KIT, which is located on the surface of HSCs. Membrane-bound SCF is a more potent stimulator of KIT, and it supports the retention of HSCs in the microenvironment via activation of VLA4 and VLA5 integrins on the HSC to promote adhesion. Membrane-bound SCF is also essential to osteoblast proliferation and the maintenance of long-term HSC activity (Wilson and Trump 2006). Calcium gradients were also recently described as an important factor in the interaction between the niche and resident HSCs (Adams et al. 2006). Cells bearing a calcium receptor (CaR), including HSCs, respond to this local gradient. Interestingly, CaR-/- mice display hypocellularity of the bone marrow with a disproportionately small number of HSCs, but no defect in differentiation. Moreover, paratrabecular hematopoietic clusters seen in wild-type mice are absent in CaR-'- mice, and the primitive HSCs are found in extramedullary locations. In competitive transplant studies (Adams et al. 2006) HSCs from these mice exhibited a severe competitive disadvantage, indicating that the defect is cell-autonomous and may be attributable to altered homing capacity or diminished retention in the niche. Additional experiments in the same study showed that CaR-'- cells could not adhere to collagen I (the most prominent extracellular matrix protein at the endosteal surface of bone), supporting a potential role for CaR in the retention and lodgment of HSCs in the proper microenvironment. These observations indicate that the local concentration of calcium ions at the endosteal niche is important in the regulation of the HSC population (Adams et al. 2006).

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